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Purpose Hyperpolarized 13C magnetic resonance allows for the study of real-time metabolism in vivo, including significant hyperpolarized 13C lactate production in many tumors. Other studies have shown that aggressive and highly metastatic tumors rapidly transport lactate out of cells. Thus, the ability to not only measure the production of hyperpolarized 13C lactate but also understand its compartmentalization using diffusion-weighted MR will provide unique information for improved tumor characterization. Methods We used a bipolar, pulsed-gradient, double spin echo imaging sequence to rapidly generate diffusion-weighted images of hyperpolarized 13C metabolites. Our methodology included a simultaneously acquired B1 map to improve apparent diffusion coefficient (ADC) accuracy and a diffusion-compensated variable flip angle scheme to improve ADC precision. Results We validated this sequence and methodology in hyperpolarized 13C phantoms. Next, we generated ADC maps of several hyperpolarized 13C metabolites in a normal rat, rat brain tumor, and prostate cancer mouse model using both preclinical and clinical trial-ready hardware. Conclusion ADC maps of hyperpolarized 13C metabolites provide information about the localization of these molecules in the tissue microenvironment. The methodology presented here allows for further studies to investigate ADC changes due to disease state that may provide unique information about cancer aggressiveness and metastatic potential. Magn Reson Med 74:622–633, 2015. © 2014 Wiley Periodicals, Inc.

The prognosis for patients with pancreatic cancer is extremely poor, as evidenced by the disease's five-year survival rate of ~5%. New approaches are therefore urgently needed to improve detection, treatment, and monitoring of pancreatic cancer. MRS-detectable metabolic changes provide useful biomarkers for tumor detection and response-monitoring in other cancers. The goal of this study was to identify MRS-detectable biomarkers of pancreatic cancer that could enhance currently available imaging approaches. We used (1) H high-resolution magic angle spinning MRS to probe metabolite levels in pancreatic tissue samples from mouse models and patients. In mice, the levels of lipids dropped significantly in pancreata with lipopolysaccharide-induced inflammation, in pancreata with pre-cancerous metaplasia (4 week old p48-Cre;LSL-Kras(G12D) mice), and in pancreata with pancreatic intraepithelial neoplasia, which precedes invasive pancreatic cancer (8 week old p48-Cre LSL-Kras(G12D) mice), to 26 ± 19% (p = 0.03), 19 ± 16% (p = 0.04), and 26 ± 10% (p = 0.05) of controls, respectively. Lactate and taurine remained unchanged in inflammation and in pre-cancerous metaplasia but increased significantly in pancreatic intraepithelial neoplasia to 266 ± 61% (p = 0.0001) and 999 ± 174% (p < 0.00001) of controls, respectively. Importantly, analysis of patient biopsies was consistent with the mouse findings. Lipids dropped in pancreatitis and in invasive cancer biopsies to 29 ± 15% (p = 0.01) and 26 ± 38% (p = 0.02) of normal tissue. In addition, lactate and taurine levels remained unchanged in inflammation but rose in tumor samples to 244 ± 155% (p = 0.02) and 188 ± 67% (p = 0.02), respectively, compared with normal tissue. Based on these findings, we propose that a drop in lipid levels could serve to inform on pancreatitis and cancer-associated inflammation, whereas elevated lactate and taurine could serve to identify the presence of pancreatic intraepithelial neoplasia and invasive tumor. Our findings may help enhance current imaging methods to improve early pancreatic cancer detection and monitoring.

Metabolic reprogramming is increasingly being viewed as a hallmark of cancer. Accordingly, metabolic readouts can serve as biomarkers of response to therapy. The goal of this study was to investigate some of the MRS-detectable metabolic consequences of mitogen-activated protein kinase kinase (MEK) inhibition. We investigated PC3 prostate cancer, MCF-7 breast cancer and A375 melanoma cells, and determined that, consistent with previous studies, MRS-detectable levels of phosphocholine decreased significantly in all cell lines (to 63%, 50% and 18% of the control, respectively) following MEK inhibition with U0126. This effect was mediated by a decrease in the expression of choline kinase α, the enzyme that catalyzes the phosphorylation of choline. In contrast, the impact of MEK inhibition on glycolysis was cell line dependent. A375 cells, which express mutant BRAF, demonstrated significant decreases in glucose uptake (to 36% of control) and lactate production (to 42% of control) in line with positron emission tomography data. In contrast, in PC3 and MCF-7 cells, increases in glucose uptake (to 198% and 192% of control, respectively) and lactate production (to 177% and 212% of control, respectively) were observed, in line with a previous hyperpolarized (13) C MRS study. This effect is probably mediated by the activation of the phosphoinositide 3-kinase pathway and AMP-activated protein kinase. Our findings demonstrate the value of translatable non-invasive MRS methods for the provision of information on cellular metabolism as an indication of the activation of potential feedback loops following MEK inhibition.

Low-grade gliomas, driven by mutations in the isocitrate dehydrogenase 1 (IDH1) gene, are less aggressive than primary glioblastoma (GBM), but nonetheless always recur and ultimately lead to patient death. To improve patient survival, one therapeutic strategy is treatment with the alkylating chemotherapeutic agent Temozolomide (TMZ), previously reserved for the treatment of primary GBM. Though the treatment of IDH1 mutant patients with TMZ improves survival, early detection of treatment-associated effects remains challenging. The goal of this study was, therefore, to determine the value of magnetic resonance spectroscopy (MRS)-based biomarkers for detection of response to treatment. To this end, we examined the global metabolic alterations that occurred following TMZ treatment in a genetically engineered IDH1 mutant immortalized Normal Human Astrocyte (NHAIDHmut)-based cell model using 1H MRS combined with chemometrics and 13C MRS combined with labeling of cells with [1-13C]-glucose and [3-13C]-glutamine. Cells were treated either with the IC50 value of TMZ (100 μM; N=5), or with DMSO (0.2%; N=5) for 72 hours. Then, metabolites were extracted from cells using the dual-phase extraction method. 1H-MRS and proton-decoupled 13C-MRS spectra were acquired using a 500 MHz Bruker Avance spectrometer. 1H MRS data was subjected to multivariate analysis using SIMCA. Specific metabolites were also quantified using Mnova7, integrals normalized to TSP and to cell number and statistical significance of differences determined using unpaired Student’s t-test. Pathway enrichment analysis of dysregulated metabolites was performed using MetPA. principal component analysis score plot showed separation of TMZ-treated from DMSO-treated control cells. Further, improved separation between the groups was obtained by orthogonal-partial least squares discriminant analysis. Most significant metabolites contributing to class separation were identified using correlation ≥0.6 and variable importance in projection score ≥1. Glutamine, glutamate, 2-hydroxyglutarate (2-HG), pyruvate, succinate, glucose, phosphocholine, isoleucine, valine, lysine, phenylalanine, NAD(P)+ and AMP/ADP/ATP were observed to be significantly higher in TMZ-treated cells as compared to controls. Based on pathway impact score, tricarboxylic acid (TCA) cycle was identified to be the significantly altered pathway following treatment Further, there was a significant increases in glucose- and glutamine-derived glutamate and 2-HG, indicating an increase in flux to the TCA cycle following treatment. These findings demonstrate that IDH1 mutant glioma show a clear metabolomic fingerprint in response to TMZ therapy, which, combined with complementary hyperpolarized 13C MRS approaches, may help assess early response to TMZ therapy in mutant IDH1 glioma. Note: This abstract was not presented at the meeting. Citation Format: Elavarasan Subramani, Chloe Najac, Georgios Batsios, Pavithra Viswanath, Marina Radoul, Anne Marie Gillespie, Russell O. Pieper, Sabrina M. Ronen. 1H and 13C MRS-based metabolic markers of IDH1 mutant glioma response to temozolomide therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5263.

Vorinostat is a histone deacetylase (HDAC) inhibitor that inhibits cell proliferation and induces apoptosis in solid tumors, and is in clinical trials for the treatment of glioblastoma (GBM). The goal of this study was to assess whether hyperpolarized 13 C MRS and magnetic resonance spectroscopic imaging (MRSI) can detect HDAC inhibition in GBM models. First, we confirmed HDAC inhibition in U87 GBM cells and evaluated real-time dynamic metabolic changes using a bioreactor system with live vorinostat-treated or control cells. We found a significant 40% decrease in the 13 C MRS-detectable ratio of hyperpolarized [1-13 C]lactate to hyperpolarized [1-13 C]pyruvate, [1-13 C]Lac/Pyr, and a 37% decrease in the pseudo-rate constant, kPL , for hyperpolarized [1-13 C]lactate production, in vorinostat-treated cells compared with controls. To understand the underlying mechanism for this finding, we assessed the expression and activity of lactate dehydrogenase (LDH) (which catalyzes the pyruvate to lactate conversion), its associated cofactor nicotinamide adenine dinucleotide, the expression of monocarboxylate transporters (MCTs) MCT1 and MCT4 (which shuttle pyruvate and lactate in and out of the cell) and intracellular lactate levels. We found that the most likely explanation for our finding that hyperpolarized lactate is reduced in treated cells is a 30% reduction in intracellular lactate levels that occurs as a result of increased expression of both MCT1 and MCT4 in vorinostat-treated cells. In vivo 13 C MRSI studies of orthotopic tumors in mice also showed a significant 52% decrease in hyperpolarized [1-13 C]Lac/Pyr when comparing vorinostat-treated U87 GBM tumors with controls, and, as in the cell studies, this metabolic finding was associated with increased MCT1 and MCT4 expression in HDAC-inhibited tumors. Thus, the 13 C MRSI-detectable decrease in hyperpolarized [1-13 C]lactate production could serve as a biomarker of response to HDAC inhibitors.

Mutations in the isocitrate dehydrogenase gene IDH1 are common in low-grade glioma, where they result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, and gliomagenesis. IDH1 mutations also cosegregate with mutations in the ATRX gene and the TERT promoter, suggesting that IDH mutation may drive the creation or selection of telomere-stabilizing events as part of immortalization/transformation process. To determine whether and how this may occur, we investigated the phenotype of pRb-/p53-deficient human astrocytes engineered with IDH1 wild-type (WT) or R132H-mutant (IDH1mut) genes as they progressed through their lifespan. IDH1mut expression promoted 2HG production and altered histone methylation within 20 population doublings (PD) but had no effect on telomerase expression or telomere length. Accordingly, cells expressing either IDH1WT or IDH1mut entered a telomere-induced crisis at PD 70. In contrast, only IDH1mut cells emerged from crisis, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo. Clonal populations of postcrisis IDH1mut cells displayed shared genetic alterations, but no mutations in ATRX or the TERT promoter were detected. Instead, these cells reactivated telomerase and stabilized their telomeres in association with increased histone lysine methylation (H3K4me3) and c-Myc/Max binding at the TERT promoter. Overall, these results show that although IDH1mut does not create or select for ATRX or TERT promoter mutations, it can indirectly reactivate TERT, and in doing so contribute to astrocytic immortalization and transformation. Cancer Res; 76(22); 6680–9. ©2016 AACR.

Myeloid-derived suppressor cells (MDSCs) are highly prevalent inflammatory cells that play a key role in tumor development and are considered therapeutic targets. MDSCs promote tumor growth by blocking T-cell-mediated anti-tumoral immune response through depletion of arginine that is essential for T-cell proliferation. To deplete arginine, MDSCs express high levels of arginase, which catalyzes the breakdown of arginine into urea and ornithine. Here, we developed a new hyperpolarized 13C probe, [6-13C]-arginine, to image arginase activity. We show that [6-13C]-arginine can be hyperpolarized and hyperpolarized [13C]-urea production from [6-13C]-arginine is linearly correlated with arginase concentration in vitro. Furthermore we show that we can detect a statistically significant increase in hyperpolarized [13C]-urea production in MDSCs when compared to control bone marrow cells. This increase was associated with an increase in intracellular arginase concentration detected using a spectrophotometric assay. Hyperpolarized [6-13C]-arginine could therefore serve to image tumoral MDSC function and more broadly M2-like macrophages.

The current standard of care for glioblastoma (GBM) is surgical resection, radiotherapy, and treatment with temozolomide (TMZ). However, resistance to current therapies and recurrence are common. To improve survival, agents that target the PI3K signaling pathway, which is activated in approximately 88% of GBM, are currently in clinical trials. A challenge with such therapies is that tumor shrinkage is not always observed. New imaging methods are therefore needed to monitor response to therapy and predict survival. The goal of this study was to determine whether hyperpolarized 13C magnetic resonance spectroscopic imaging (MRSI) and 1H magnetic resonance spectroscopy (MRS) can be used to monitor response to the second-generation dual PI3K/mTOR inhibitor voxtalisib (XL765, SAR245409), alone or in combination with TMZ. We investigated GS-2 and U87-MG GBM orthotopic tumors in mice, and used MRI, hyperpolarized 13C MRSI, and 1H MRS to monitor the effects of treatment. In our study, 1H MRS could not predict tumor response to therapy. However, in both our models, we observed a significantly lower hyperpolarized lactate-to-pyruvate ratio in animals treated with voxtalisib, TMZ, or combination therapy, when compared with controls. This metabolic alteration was observed prior to MRI-detectable changes in tumor size, was consistent with drug action, and was associated with enhanced animal survival. Our findings confirm the potential translational value of the hyperpolarized lactate-to-pyruvate ratio as a biomarker for noninvasively assessing the effects of emerging therapies for patients with GBM. Mol Cancer Ther; 15(5); 1113–22. ©2016 AACR.

Mutations in isocitrate dehydrogenase 1 (IDH1) characterize most adult low-grade gliomas. Mutant IDH1 catalyzes production of the oncometabolite 2-hydroxyglutarate (2-HG). We recently discovered that the IDH1 mutation also reprograms pyruvate metabolism in a 2-HG-dependent manner, and that reprogramming of pyruvate metabolism is essential for cell proliferation in glioma cells with mutant IDH1.

Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG). Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG). In turn, 2-HG, which has been termed an "oncometabolite," inhibits key α-KG-dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprograming that extends beyond 2-HG production, and this reprograming often differs from what has been previously reported in other cancer types. In this review, we will discuss in detail what is known to date about the metabolic reprograming of mutant IDH cells, and how this reprograming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells, and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

To develop a compressed sensing (CS) acceleration method with a high spectral bandwidth exploiting the spatial-spectral sparsity of MR spectroscopic imaging (MRSI).Accelerations were achieved using blip gradients during the readout to perform nonoverlapped and stochastically delayed random walks in kx -ky -t space, combined with block-Hankel matrix completion for efficient reconstruction. Both retrospective and prospective CS accelerations were applied to (13) C MRSI experiments, including in vivo rodent brain and liver studies with administrations of hyperpolarized [1-(13) C] pyruvate at 7.0 Tesla (T) and [2-(13) C] dihydroxyacetone at 3.0 T, respectively.In retrospective undersampling experiments using in vivo 7.0 T data, the proposed method preserved spectral, spatial, and dynamic fidelities with R(2) ≥ 0.96 and ≥ 0.87 for pyruvate and lactate signals, respectively, 750-Hz spectral separation, and up to 6.6-fold accelerations. In prospective in vivo experiments, with 3.8-fold acceleration, the proposed method exhibited excellent spatial localization of metabolites and peak recovery for pyruvate and lactate at 7.0 T as well as for dihydroxyacetone and its metabolic products with a 4.5-kHz spectral span (140 ppm at 3.0 T).This study demonstrated the feasibility of a new CS approach to accelerate high spectral bandwidth MRSI experiments. Magn Reson Med 76:369-379, 2016. © 2016 Wiley Periodicals, Inc.

In vivo spin spin relaxation time (T2) heterogeneity of hyperpolarized [(13)C,(15)N2]urea in the rat kidney was investigated. Selective quenching of the vascular hyperpolarized (13)C signal with a macromolecular relaxation agent revealed that a long-T2 component of the [(13)C,(15)N2]urea signal originated from the renal extravascular space, thus allowing the vascular and renal filtrate contrast agent pools of the [(13)C,(15)N2]urea to be distinguished via multi-exponential analysis. The T2 response to induced diuresis and antidiuresis was performed with two imaging agents: hyperpolarized [(13)C,(15)N2]urea and a control agent hyperpolarized bis-1,1-(hydroxymethyl)-1-(13)C-cyclopropane-(2)H8. Large T2 increases in the inner-medullar and papilla were observed with the former agent and not the latter during antidiuresis. Therefore, [(13)C,(15)N2]urea relaxometry is sensitive to two steps of the renal urea handling process: glomerular filtration and the inner-medullary urea transporter (UT)-A1 and UT-A3 mediated urea concentrating process. Simple motion correction and subspace denoising algorithms are presented to aid in the multi exponential data analysis. Furthermore, a T2-edited, ultra long echo time sequence was developed for sub-2 mm(3) resolution 3D encoding of urea by exploiting relaxation differences in the vascular and filtrate pools.

Alterations in cell metabolism are increasingly recognized as a hallmark of cancer and are being exploited for the development of diagnostic tools and targeted therapeutics. Recently, 13C magnetic resonance spectroscopy (MRS)-detectable hyperpolarized pyruvate to lactate conversion was validated in models as a noninvasive imaging method for the detection of tumors and treatment response, and successfully passed phase I clinical trials. To date, response to treatment was associated with a drop in hyperpolarized lactate production. Here, we monitored the effect of treatment with the MEK inhibitor U0126 in prostate and breast cancer cells. Following treatment we observed a 31% drop in flux of hyperpolarized 13C label in treated MCF-7 breast cancer cells compared to controls. In contrast and unexpectedly, flux increased to 167% in treated PC3 prostate cancer cells. To mechanistically explain these observations we investigated treatment-induced changes in the different factors known to affect pyruvate to lactate conversion. NADH levels remained unchanged whereas lactate dehydrogenase expression and activity as well as intracellular lactate increased in both cell lines, providing an explanation for the elevated hyperpolarized lactate observed in PC3 cells. The expression of MCT1, which mediates pyruvate transport, dropped in treated MCF-7 but not in PC3 cells. This identifies pyruvate transport as rate limiting in U0126-treated MCF-7 cells and explains the drop in hyperpolarized lactate observed in those cells following treatment. Our findings highlight the complexity of interactions between MEK and metabolism, and the need for mechanistic validation before hyperpolarized 13C MRS can be used for monitoring treatment-induced molecular responses.

Dynamic nuclear polarization and the recent development of a dissolution process that retains polarization in liquid state have allowed for the acquisition of 13C magnetic resonance spectroscopic imaging (MRSI) data with a substantial gain in sensitivity over conventional MR methods (1). Recent studies using hyperpolarized [1-13C]-pyruvate as a substrate have demonstrated the promise of this technique for examining in vivo tumor metabolism in brain tumors (2–4). These preclinical studies have shown the feasibility of using this technique for differentiating tumor from normal brain tissue and detecting an early response to treatment in animal models of high-grade gliomas. The further refinement of 13C MRSI acquisition techniques with higher spatial resolution is of interest for assessing spatial variations in tissue metabolism due to the infiltrative nature and molecular heterogeneity of gliomas. One of the major obstacles in acquiring hyperpolarized 13C signal is the time limitation imposed by the T1 decay of hyperpolarized compound. In order to more efficiently sample the rapidly decaying 13C signal, fast imaging techniques, such as compressed sensing, have been incorporated in the acquisition of hyperpolarized 13C data (5). By taking advantage of the sparsity in hyperpolarized 13C spectra and the undersampling of spectral k-space, the compressed sensing technique has enabled the acquisition of hyperpolarized 13C metabolites with high temporal and spatial resolution in preclinical studies involving transgenic prostate cancer and liver cancer murine models (5,6). The purpose of this project was to implement a compressed sensing scheme tailored to the rat brain morphology and to demonstrate the utility of this method in obtaining hyperpolarized 13C MRSI data for evaluating heterogeneous 13C metabolic profiles within brain tumor tissue using an orthotopic human glioblastoma (GBM) xenograft model. The new compressed sensing sampling scheme was validated through phantom experiments and by comparing with fully sampled 13C 3D MRSI data. Our emphasis was to quantitatively assess the compressed sensing hyperpolarized 13C MRSI data and to compare 13C metabolic profiles from different regions of tumor with the results from histology and immunohistochemistry.

IDH1 mutations in lower grade glioma result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, altered gene expression, and cellular transformation. IDH1 mutations, however, are also linked to mutations in the ATRX gene and the hTERT promoter, suggesting that IDH1 mutation contributes to events that stabilize telomeres and help drive the cellular immortalization and transformation processes. To determine if and how IDH1 mutation influences telomeric stabilization and cellular immortalization, pRb/p53-deficient normal human astrocytes (E6/E7 NHA) were lentivirally infected with constructs encoding WT (IDH1 WT) or R132H mutant IDH 1 (IDH1mut). Following selection, the cells were then monitored for 2HG levels, histone modifications, hTERT promoter mutations, ATRX and telomerase expression, and telomere length as they progressed through their lifespan. Introduction of IDH1mut, but not IDH1 WT, resulted in 2HG production and alterations in histone methylation within 20 population doublings (PD). Neither IDH1mut nor IDH1 WT expression, however, had any direct effect on telomerase expression or telomere length, and cells expressing either IDH1 WT or IDH1mut entered a telomere-induced crisis at PD 70. While cells expressing IDH1 WT failed to reactivate telomerase and escape from crisis, cells expressing IDH1mut emerged from crisis after 51 days, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo. The transformed IDH1mut cells did not exhibit the ALT phenotype or hTERT promoter mutations, but rather exhibited telomerase expression and activity, and shortened but stabilized telomeres. These results show that while IDH1 mutations do not directly contribute to ATRX gene mutation, hTERT promoter mutation, or TERT reactivation, they are permissive for the events that indirectly drive the TERT reactivation that contributes to both the immortalization and transformation of astrocytic cells.

Introduction: Missense R132H mutations in the active site of isocitrate dehydrogenase 1 (IDH1) biologically and diagnostically distinguish low-grade gliomas and secondary glioblastomas from primary glioblastomas. IDH1 mutations lead to the formation of the oncometabolite 2-hydroxyglutarate (2-HG) from the reduction of α-ketoglutarate (α-KG), which in turn facilitates tumorigenesis by modifying DNA and histone methylation as well blocking differentiation processes. We recently showed (Mol Cancer Res 14: 976–983, 2016) that although mutant IDH1 expression in hTERT-immortalized, p53/pRb-deficient astrocytes can drive cellular transformation and gliomagenesis, selective pharmacologic inhibition and elimination of 2-HG by the mutant IDH1 inhibitor AGI-5198 has little effect on the growth or clonagenicity of these transformed cells. To address the possible role of WT IDH1 in the growth of mutant IDH-driven tumor cells, we used a slightly different gliomagenesis model in which the transformation of TERT-deficient, p53/pRb-deficient astrocytes (pre-crisis cells) occurs only after prolonged expression of mutant IDH and passage through cellular crisis (post-crisis cells, Cancer Res 76:6680–6689, 2016). METHODS AND MATERIALS: Using this system we introduced AGI-5198, or siRNA targeting both WT and mutant forms of IDH1 into p53/pRb-deficient, mutant IDH1-expressing human astrocytes prior to or following their transformation, and compared the effects on cell growth and clonagenicity. Results: AGI-5198 exposure decreased levels of 2HG by greater than 90%, and as previously reported had no effect on the growth of either the pre-or post-crisis cell populations. A one-day exposure to a pan IDH1 siRNA resulted in a similar, prolonged (greater than 6 day), 80% inhibition of both WT and mutant IDH1 protein levels and 2HG in both cell groups. While the growth of the mutant IDH-expressing, non-transformed cells was similar to that of scramble siRNA controls, the growth of the mutant IDH-transformed cells was significantly reduced. This growth suppression was also accompanied by a four-fold increase in annexin V-positive apoptotic cells. Furthermore, the growth suppression in the cells transformed by mutant IDH1 expression could not be reversed by addition of a cell-permeable form of 2-HG. Conclusions: These results show that the in vitro transformative events driven by expression of mutant IDH1 make cells dependent not on continued mutant IDH1 expression, but rather on continued WT IDH1 expression. The data also support the development and testing of agents that can inhibit both the WT and mutant forms of IDH1.

The phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway is activated in more than88% of glioblastomas (GBM). New drugs targeting this pathway are currently in clinical trials. However, noninvasive assessment of treatment response remains challenging. By using magnetic resonance spectroscopy (MRS), PI3K/Akt/mTOR pathway inhibition was monitored in 3 GBM cell lines (GS-2, GBM8, and GBM6; each with a distinct pathway activating mutation) through the measurement of 2 mechanistically linked MR biomarkers: phosphocholine (PC) and hyperpolarized lactate.(31)P MRS studies showed that treatment with the PI3K inhibitor LY294002 induced significant decreases in PC to 34 %± 9% of control in GS-2 cells, 48% ± 5% in GBM8, and 45% ± 4% in GBM6. The mTOR inhibitor everolimus also induced a significant decrease in PC to 62% ± 14%, 57% ± 1%, and 58% ± 1% in GS-2, GBM8, and GBM6 cells, respectively. Using hyperpolarized (13)C MRS, we demonstrated that hyperpolarized lactate levels were significantly decreased following PI3K/Akt/mTOR pathway inhibition in all 3 cell lines to 51% ± 10%, 62% ± 3%, and 58% ± 2% of control with LY294002 and 72% ± 3%, 61% ± 2%, and 66% ± 3% of control with everolimus in GS-2, GBM8, and GBM6 cells, respectively. These effects were mediated by decreases in the activity and expression of choline kinase α and lactate dehydrogenase, which respectively control PC and lactate production downstream of HIF-1. Treatment with the DNA damaging agent temozolomide did not have an effect on either biomarker in any cell line. This study highlights the potential of PC and hyperpolarized lactate as noninvasive MR biomarkers of response to targeted inhibitors in GBM.

Bone metastasis is a major cause of morbidity and mortality in prostate cancer. However, the lack of clinically relevant models hinders our understanding of the disease as well as development of effective therapies and imaging approaches. We used noninvasive MRI, histology and micro CT to further characterize the newly established prostate cancer bone metastases-derived model MDA-PCa-118b, and to compare it to the well-established PC-3MM2 model with regard to bone structure and vascular patterning. The PC-3MM2 model is highly osteolytic whereas the MDA-PCa-118b model shows a robust osteoblastic reaction, as often seen in clinical cases. Macromolecular contrast enhanced MRI revealed differences in vascular permeability patterns, which appeared peripheral for PC-3MM2 and nodular for MDA-PCa-118b, matching the microscopic cellular composition of each model: PC-3MM2 exclusively recruits endothelial cells to form thin tumor-core blood vessels and enlarged, leaky peripheral vessels, whereas MDA-PCa-118b also recruits bone-forming cells and pericytes such that small tumor nests are encircled with leaky vessels and embedded in bone-like tissue dotted with pericyte-covered vessels. Despite these structural differences, vascular permeability was reduced in both tumor models by either imatinib or SU10944 treatment. This study highlights the importance of clinically relevant osteogenic models of human prostate cancer and the value of such models not only in enhancing our understanding of tumorigenesis, metastasis and response to therapy, but also for development of appropriate methods for noninvasive imaging of these processes. Copyright © 2011 John Wiley & Sons, Ltd.

Abnormal choline metabolism is emerging as a metabolic hallmark that is associated with oncogenesis and tumour progression. Following transformation, the modulation of enzymes that control anabolic and catabolic pathways causes increased levels of choline-containing precursors and breakdown products of membrane phospholipids. These increased levels are associated with proliferation, and recent studies emphasize the complex reciprocal interactions between oncogenic signalling and choline metabolism. Because choline-containing compounds are detected by non-invasive magnetic resonance spectroscopy (MRS), increased levels of these compounds provide a non-invasive biomarker of transformation, staging and response to therapy. Furthermore, enzymes of choline metabolism, such as choline kinase, present novel targets for image-guided cancer therapy.

Activation of the PI3K/Akt pathway is associated with the development of numerous human cancers. As a result, many emerging therapies target this pathway. Previous studies have shown that targeting the PI3K/Akt pathway at the level of PI3K is associated with a drop in phosphocholine (PCho) and a reduction in hyperpolarized lactate production. However, the consequences of targeting downstream of PI3K at the level of Akt have not been investigated. Perifosine is an anticancer alkylphospholipid used in clinical trials. It acts by inhibiting phosphorylation of Akt and has been shown to inhibit CTP-phosphocholine cytidyltransferase (CT). The goal of this study was to identify the MRS-detectable metabolic consequences of treatment with perifosine in MCF-7 breast cancer cells. We found that perifosine treatment led to a 51 ± 5% drop in PCho from 30 ± 5 to 15 ± 1 fmol/cell and a comparable drop in de novo synthesized PCho. This was associated with a drop in choline kinase (ChoK) activity and ChoKα expression. CT inhibition could not be ruled out but likely did not contribute to the change in PCho. We also found that intracellular lactate levels decreased from 2.7 ± 0.5 to 1.5 ± 0.3 fmol/cell and extracellular lactate levels dropped by a similar extent. These findings were consistent with a drop in lactate dehydrogenase expression and associated with a drop in activity of the hypoxia inducible factor (HIF)-1α. The drops in PCho and lactate production following perifosine treatment are therefore mediated downstream of Akt by the drop in HIF-1α, which serves as the transcription factor for both ChoK and lactate dehydrogenase. The metabolic changes were confirmed in a second breast cancer cell line, MDA-MB-231. Taken together, these findings indicate that PCho and lactate can serve as noninvasive metabolic biomarkers for monitoring the effects of inhibitors that target the PI3K/Akt pathway, independent of the step that leads to inhibition of HIF-1α. Copyright © 2011 John Wiley & Sons, Ltd.

The general course of treatment for patients with brain tumors consists of surgery, radiation therapy, and chemotherapy with the goal of stopping tumor growth by inhibiting cell proliferation or inducing cell death. Temozolomide (TMZ) is one of the most frequently used chemotherapeutic drugs for treating brain tumors, especially high-grade gliomas. It is an alkylating agent and works by damaging DNA and thus triggering the death of tumor cells. The current standard for monitoring brain tumor response to therapy is a combination of clinical symptoms and Macdonald criteria, which are based on the change in contrast enhancement from magnetic resonance imaging (MRI) or computed tomography (CT) scans (1). Advanced MR techniques such as 1H magnetic resonance spectroscopy (MRS), perfusion MRI, and diffusion-weighted MRI have also been used for monitoring brain tumor response to therapy (2–7), but may take several weeks to detect response to therapy. Early detection of brain tumor response to treatment would be valuable in defining criteria for continuing or modifying treatment strategies. Positron emission tomography (PET) is an imaging modality that has been widely used in the clinic for predicting treatment effects. Although the ability to detect altered 18F-fluoro-dexoyglucose (FDG) uptake in cancerous tissue has made this technique useful in some areas of oncology (8), its use in neuro-oncology presents unique challenges due to the high background signal from normal gray matter (9). Dynamic nuclear polarization (DNP) and the recent development of a dissolution process that retains polarization in liquid state have allowed for the acquisition of 13C MRS data with more than 10,000-fold increase in sensitivity (10). Preclinical 13C MRS of hyperpolarized [1-13C]-pyruvate has been applied to the investigation of in vivo metabolism in subcutaneous lymphoma (11), prostate cancer (12,13), cardiac ischemia (14), and liver cancer (15). A recent study has demonstrated that this technique can be used for differentiating brain tumors from normal tissue in an animal model of brain cancer (16). The purpose of this study was to demonstrate the feasibility of using DNP-hyperpolarized [1-13C]-pyruvate to measure response to chemotherapy using TMZ in an orthotopic human glioblastoma xenograft model. Our emphasis was to establish the time frame of changes in 13C imaging parameters and to compare the results with alterations in tumor volume over time.

Tumorigenesis and metastasis are associated with metabolic reprogramming, including enhanced glycolysis and glutaminolysis. Recent developments in dynamic nuclear polarization (DNP) dramatically enhance the sensitivity of magnetic resonance spectroscopy, providing a novel method for metabolic imaging in real-time through 'hypersensing' of polarized metabolites. In parallel, advanced signal acquisition sequences, such as compressed sensing, improve spatial and temporal resolution providing better depiction of rapid metabolic events in small tumors and metastases. Here we review the potential of hyperpolarized magnetic resonance spectroscopy for studying the crosstalk between oncogenic cell signaling and metabolism, and for probing diagnostic biomarkers and metabolic indicators of therapeutic response in primary tumors and metastases.

Introduction: Cancer cells up-regulate phospholipid biosynthesis in order to meet the demands of uncontrolled cell proliferation. Unusually, mutant isocitrate dehydrogenase 1 (IDHmut) gliomas down-regulate levels of the phospholipid precursors phosphocholine and phosphoethanolamine relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was therefore to investigate whether the phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdE) are also down-regulated in IDHmut gliomas. Methods: Immortalized normal human astrocytes and U87 cells expressing IDHwt or IDHmut enzyme were studied. Steady-state phospholipid levels were measured by 31P-magnetic resonance spectroscopy (MRS) of cell extracts. De novo phospholipid biosynthesis was quantified by 13C-MRS of cells labeled with 56μM [1,2-13C]-choline and [1,2-13C]-ethanolamine. ER area was measured by STORM imaging of calnexin fluorescence. Results: Steady-state PtdCho and PtdE levels and de novo phospholipid biosynthesis were significantly reduced in IDHmut cells relative to IDHwt. Concomitantly, the activities of CTP:PC cytidylyltransferase (CCT) and CTP:PE cytidylyltransferase (ECT), the rate-limiting enzymes for phospholipid biosynthesis, were significantly reduced in IDHmut cells. CCT and ECT are localized to the endoplasmic reticulum (ER), the site of phospholipid biosynthesis. We therefore measured ER area by super-resolution STORM imaging and found a significant reduction in IDHmut cells relative to IDHwt while cell size remained unchanged. Autophagy of the ER (ER-phagy) can regulate ER area and we found that IDHmut cells showed increased expression of FAM134b, the ER-phagy receptor and higher autophagic flux. These results suggest that IDHmut cells undergo ER-phagy, leading to reduced phospholipid biosynthesis. Silencing FAM134b or the autophagy proteins Atg5 and Atg7 restored ER area and phospholipid biosynthesis and abrogated clonogenicity of IDHmut cells, linking ER-phagy to reduced phospholipid biosynthesis and pointing to a therapeutic opportunity. In the absence of specific ER-phagy inhibitors, we used the late-stage autophagy inhibitor chloroquine (CQ) to test proof-of-principle and found that CQ significantly inhibited orthotopic IDHmut glioma growth in vivo. Importantly, we observed ER-phagy and down-regulation of phospholipid levels in IDHmut glioma patient biopsies, highlighting the translational validity of our findings. Conclusions: We find that down-regulation of phospholipid biosynthesis is a unique metabolic outcome of ER-phagy in IDHmut gliomas. While autophagy has been linked to glucose and glutamine metabolism, this study is the first to link autophagy to phospholipid biosynthesis. Importantly, our study identifies a rationale for the development of novel therapies targeting ER-phagy in IDHmut gliomas. Citation Format: Pavithra Viswanath, Russell O. Pieper, Joanna J. Phillips, Sabrina M. Ronen. Autophagic degradation of the endoplasmic reticulum leads to reduced phospholipid biosynthesis in mutant isocitrate dehydrogenase 1 gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1431.

The phosphatidylinositol-3-kinase (PI3K)/Akt oncogenic pathway is critical in glioblastomas. Loss of PTEN, a negative regulator of the PI3K pathway or activated PI3K/Akt pathway that drive increased proliferation, survival, neovascularization, glycolysis, and invasion is found in 70%-80% of malignant gliomas. Thus, PI3K is an attractive therapeutic target for malignant glioma. We report that a new irreversible PI3K inhibitor, PX-866, shows potent inhibitory effects on the PI3K/Akt signaling pathway in glioblastoma. PX-866 did not induce any apoptosis in glioma cells; however, an increase in autophagy was observed. PX-866 inhibited the invasive and angiogenic capabilities of cultured glioblastoma cells. In vivo, PX-866 inhibited subcutaneous tumor growth and increased the median survival time of animals with intracranial tumors. We also assessed the potential of proton magnetic resonance spectroscopy (MRS) as a noninvasive method to monitor response to PX-866. Our findings show that PX-866 treatment causes a drop in the MRS-detectable choline-to-NAA, ratio and identify this partial normalization of the tumor metabolic profile as a biomarker of molecular drug action. Our studies affirm that the PI3K pathway is a highly specific molecular target for therapies for glioblastoma and other cancers with aberrant PI3K/PTEN expression.

Numerous mechanism-based anticancer drugs that target the phosphatidylinositol-3-kinase (PI3K) signaling pathway are in clinical trials. However, assessment of response using traditional imaging methods remains a challenge, as it is often associated with tumor stasis. Here we show for the first time the efficacy of hyperpolarized 13C magnetic resonance spectroscopy (MRS) in detecting the effect of PI3K signaling inhibition by monitoring hyperpolarized [1-13C]-lactate levels produced from hyperpolarized [1-13C]-pyruvate through lactate dehydrogenase (LDH) activity. Studies were performed on GS-2 glioblastoma and MDA-MB-231 breast adenocarcinoma cells. Following inhibition of signaling, hyperpolarized lactate dropped significantly to 42±12% and 76±5% in GS-2 cells treated with LY294002 and everolimus respectively and to 71±15% in MDA-MB-231 cells treated with LY294002. This correlated with a drop in LDH activity to 48±4%, 63±4% and 69±12%, and was associated with a drop in LDHA mRNA levels and LDHA and HIF-1α protein levels. Taken together the data demonstrate that hyperpolarized 13C MRS of pyruvate can be used to monitor the drop in LDHA activity and expression following reduced HIF-1α levels resulting from PI3K signal inhibition. In line with these findings, in vivo studies of GS-2 subcutaneous xenografts treated with everolimus resulted in inhibition in tumor growth, which was associated with a drop in the hyperpolarized lactate-to-pyruvate ratio. In contrast an increase in the ratio was detected in controls. This works demonstrates the potential of hyperpolarized 13C MRS for noninvasive imaging of drug target modulation by treatments that modulate PI3K signaling and HIF-1α levels.

Mutations in isocitrate dehydrogenase 1 (IDH1) drive tumorigenesis in low-grade gliomas and result in production of the oncometabolite 2-hydroxyglutarate (2-HG). 2-HG extensively reprograms cell metabolism, often in a manner atypical relative to wild-type IDH1 gliomas. In particular, compared to normal tissue, wild-type IDH1 gliomas are characterized by elevated levels of phosphocholine (PC) and phosphoethanolamine (PE), which are intermediates in the synthesis of the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. Mutant IDH1 gliomas, however, down-regulate PC and PE levels and we previously linked this effect to 2-HG-mediated down-regulation of choline kinase (CK) and ethanolamine kinase (EK) activity. The goal of this study was to delineate the molecular pathway by which 2-HG down-regulates this activity. Previous studies have linked hypoxia inducible factor-1α (HIF-1α) to modulation of CKα expression. Here, using two genetically-engineered models expressing mutant or wild-type IDH1 (U87-based and immortalized normal human astrocyte-based), we established that HIF-1α levels were higher in mutant IDH1 cells relative to wild-type. Mechanistically, silencing HIF-1α in mutant IDH1 cells restored CKα expression, CK activity, EK activity, PC and PE to levels observed in wild-type IDH1 cells. To further confirm our results, we treated mutant IDH1 cells with KC7F2, a pharmacological inhibitor of HIF-1α synthesis and found that it restored CKα expression, CK activity, EK activity, PC and PE to levels observed in wild-type cells. Importantly, HIF-1α silencing and KC7F2 treatment increased PC, PE, CKα expression, CK activity and EK activity in the clinically relevant BT54 patient-derived mutant IDH1 model. Finally, examination of orthotopic U87 tumors expressing mutant or wild-type IDH1 showed that PC, PE, CKα expression, CK activity and EK activity were reduced in mutant tumors relative to wild-type, an effect associated with higher HIF-1α levels in mutant tumors. Taken together, our study mechanistically links HIF-1α to the unusual down-regulation of phospholipid metabolism in mutant IDH1 gliomas.

Today, there is no reliable noninvasive imaging method available to monitor glioblastoma (GBM) response to therapy and predict survival prior to tumor shrinkage. Dissolution Dynamic Nuclear Polarization (DNP) combined with hyperpolarized 13C Magnetic Resonance Spectroscopic Imaging (MRSI) is a novel imaging method that allows probing real-time tumor metabolism. Recent studies using 13C MRSI have shown decreased lactate production from pyruvate in GBM responsive to a dual PI3K/mTOR inhibitor and/or Temozolomide, mediated by lower expression of LDHA or PKM2 enzymes, respectively. SAHA, the histone deacetylase (HDAC) inhibitor, is a novel drug that inhibits cell proliferation by inducing cell cycle arrest followed by apoptosis. The goal of this study was to detect early HDAC inhibition using advanced 13C MRSI. Analysis of dynamic real-time cellular metabolic changes in SAHA-treated U87 live cells in bioreactors demonstrated a significant 37.7% decrease in hyperpolarized [1-13C]-lactate production. SAHA-treated cells also showed a 29.6% decrease of steady-state lactate level in cell extracts. Furthermore, we demonstrated a significant 30.3% decrease in hyperpolarized lactate-to-pyruvate ratio in SAHA-treated U87-bearing mice, which occurred prior to MRI-detectable changes in tumor size that was associated with enhanced animal survival. In order to mechanistically validate our findings, we tested the levels of expression of LDHA, MCT1 and MCT4, the main players in pyruvate-to-lactate interconversion. While expression of LDHA, the enzyme that catalyzes pyruvate-to-lactate conversion was independent of SAHA treatment, expression of both MCT1 and MCT4 transporters, that shuttle pyruvate and lactate in and out of the cell, are increased. We thus propose that increased MCT1/4 led to a decrease in lactate in response to HDAC inhibition and resulted in a reduced pyruvate-to-lactate conversion. Our findings confirm the potential translational value of the hyperpolarized lactate-to-pyruvate ratio as a biomarker for noninvasively assessing the early effects of emerging therapies for patients with GBM.

Expression of fatty acid synthase (FASN), the key enzyme in de novo synthesis of long-chain fatty acids, is normally low but increases in cancer. Consequently, FASN is a novel target for cancer therapy. However, because FASN inhibitors can lead to tumor stasis rather than shrinkage, noninvasive methods for assessing FASN inhibition are needed. To this end, we combined 1H, 31P, and 13C magnetic resonance spectroscopy (MRS) (a) to monitor the metabolic consequences of FASN inhibition and (b) to identify MRS-detectable metabolic biomarkers of response. Treatment of PC-3 cells with the FASN inhibitor Orlistat for up to 48 h resulted in inhibition of FASN activity by 70%, correlating with 74% inhibition of fatty acid synthesis. Furthermore, we have determined that FASN inhibition results not only in lower phosphatidylcholine levels but also in a 59% drop in the phospholipid precursor phosphocholine (PCho). This drop resulted from inhibition in PCho synthesis as a result of a reduction in the cellular activity of its synthetic enzyme choline kinase. The drop in PCho levels following FASN inhibition was confirmed in SKOV-3 ovarian cancer cells treated with Orlistat and in MCF-7 breast cancer cells treated with Orlistat as well as cerulenin. Combining data from all treated cells, the drop in PCho significantly correlated with the drop in de novo synthesized fatty acid levels, identifying PCho as a potential noninvasive MRS-detectable biomarker of FASN inhibition in vivo. [Mol Cancer Ther 2008;7(8):2556–65]

First described in 2003, the dissolution dynamic nuclear polarization (DNP) technique, combined with (13)C magnetic resonance spectroscopy (MRS), has since been used in numerous metabolic studies and has become a valuable metabolic imaging method. DNP dramatically increases the level of polarization of (13)C-labeled compounds resulting in an increase in the signal-to-noise ratio (SNR) of over 50,000 fold for the MRS spectrum of hyperpolarized compounds. The high SNR enables rapid real-time detection of metabolism in cells, tissues, and in vivo. This chapter will present a comprehensive review of the DNP approaches that have been used to monitor metabolism in living systems. First, the list of (13)C DNP probes developed to date will be presented, with a particular focus on the most commonly used probe, namely [1-(13)C] pyruvate. In the next four sections, we will then describe the different factors that need to be considered when designing (13)C DNP probes for metabolic studies, conducting in vitro or in vivo hyperpolarized experiments, as well as acquiring, analyzing, and modeling hyperpolarized (13)C data.

Histone deacetylase (HDAC) inhibitors are new and promising antineoplastic agents. Current methods for monitoring early response rely on invasive biopsies or indirect blood-derived markers. Our goal was to develop a magnetic resonance spectroscopy (MRS)–based method to detect HDAC inhibition. The fluorinated lysine derivative Boc-Lys-(Tfa)-OH (BLT) was investigated as a 19F MRS molecular marker of HDAC activity together with 31P MRS of endogenous metabolites. In silico modeling of the BLT-HDAC interaction and in vitro MRS studies of BLT cleavage by HDAC confirmed BLT as a HDAC substrate. BLT did not affect cell viability or HDAC activity in PC3 prostate cancer cells. PC3 cells were treated, in the presence of BLT, with the HDAC inhibitor p-fluoro-suberoylanilide hydroxamic acid (FSAHA) over the range of 0 to 10 μmol/L, and HDAC activity and MRS spectra were monitored. Following FSAHA treatment, HDAC activity dropped, reaching 53% of control at 10 μmol/L FSAHA. In parallel, a steady increase in intracellular BLT from 14 to 32 fmol/cell was observed. BLT levels negatively correlated with HDAC activity consistent with higher levels of uncleaved BLT in cells with inhibited HDAC. Phosphocholine, detected by 31P MRS, increased from 7 to 16 fmol/cell following treatment with FSAHA and also negatively correlated with HDAC activity. Increased phosphocholine is probably due to heat shock protein 90 inhibition as indicated by depletion of client proteins. In summary, 19F MRS of BLT, combined with 31P MRS, can be used to monitor HDAC activity in cells. In principle, this could be applied in vivo to noninvasively monitor HDAC activity. [Mol Cancer Ther 2006;5(5):1325–34]

Mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade glioma and secondary glioblastoma, represent an early pathogenic event and are being considered a promising therapeutic target. Consequently, non-invasive imaging methods are needed to monitor IDH1 status. Amongst these, we previously demonstrated the use of 13C MR spectroscopic imaging of hyperpolarized [1-13C] α-ketoglutarate (α-KG) to non-invasively assess IDH1 status through the detection of the conversion of hyperpolarized α-ketoglutarate to 2-hydroxyglutarate (2-HG) catalyzed by mutant IDH1. Importantly, in addition to its oncogenic role, IDH1 mutation is also associated with global modulations in metabolism. Interestingly, a study recently uncovered a relationship between presence of IDH1 mutation and decreased activity of the branched chained amino acid transaminase 1 (BCAT1) enzyme, which transaminates amino acids while converting α-KG to glutamate. Given this new study, we decided to expand on our previous findings and investigated the potential of hyperpolarized α-KG as an imaging probe to monitor BCAT1-driven α-KG-to-glutamate conversion and its modulation in the presence of IDH1 mutation. We investigated two isogenic glioblastoma lines that differed only in their IDH1 status, and performed experiments in live cells and in vivo in rat orthotopic tumors. Following injection of hyperpolarized α-KG, hyperpolarized glutamate production was detected both in cells and in vivo, and the level of hyperpolarized glutamate was significantly lower in mutant IDH1 cells and tumors compared to their IDH1-wild-type counterparts. Importantly however, the observed drop in hyperpolarized glutamate was likely mediated not only by a drop in BCAT1 activity, but also by reductions in aspartate transaminase and glutamate dehydrogenase activities, suggesting additional metabolic reprogramming at least in our model. Hyperpolarized glutamate could thus inform on multiple mutant IDH1-associated metabolic events and serve as a secondary metabolic biomarker of IDH1 mutational status in gliomas, together with 2-HG.

Several mitogen-activated protein kinase (MAPK) signaling inhibitors are currently undergoing clinical trial as part of novel mechanism-based anticancer treatment strategies. This study was aimed at detecting biomarkers of MAPK signaling inhibition in human breast and colon carcinoma cells using magnetic resonance spectroscopy. We investigated the effect of the prototype MAPK kinase inhibitor U0126 on the 31P-MR spectra of MDA-MB-231, MCF-7 and Hs578T breast, and HCT116 colon carcinoma cells. Treatment of MDA-MB-231 cells with 50 μmol/L U0126 for 2, 4, 8, 16, 24, 32, and 40 hours caused inhibition of extracellular signal–regulated kinases (ERK1/2) phosphorylation from 2 hours onwards. 31P-MR spectra of extracted cells indicated that this was associated with a significant drop in phosphocholine levels to 78 ± 8% at 8 hours, 74 ± 8% at 16 hours, 66 ± 7% at 24 hours, 71 ± 10% at 32 hours, and 65 ± 10% at 40 hours post-treatment. In contrast, the lower concentration of 10 μmol/L U0126 for 40 hours had no significant effect on either P-ERK1/ 2 or phosphocholine levels in MDA-MB-231 cells. Depletion of P-ERK1/2 in MCF-7 and Hs578T cells with 50 μmol/L U0126 also produced a drop in phosphocholine levels to 51 ± 17% at 40 hours and 23 ± 12% at 48 hours, respectively. Similarly, in HCT116 cells, inhibition with 30 μmol/L U0126 caused depletion of P-ERK1/2 and a decrease in phosphocholine levels to 80 ± 9% at 16 hours and 61 ± 4% at 24 hours post-treatment. The reduction in phosphocholine in MDA-MB-231 and HCT116 cells correlated positively with the drop in P-ERK1/2 levels. Our results show that MAPK signaling inhibition with U0126 is associated with a time-dependent decrease in cellular phosphocholine levels. Thus, phosphocholine has potential as a noninvasive pharmacodynamic marker for monitoring MAPK signaling blockade.

Magnetic resonance spectroscopic pharmacodynamic markers of the heat shock protein 90 inhibitor 17-allylamino,17- demethoxygeldanamycin (17AAG) in human colon cancer models. Background. 17-Allylamino,17-demethoxygeldanamycin (17AAG) is a novel anticancer drug that inhibits heat shock protein 90 (Hsp90), resulting in proteasomal degradation of several oncogenic proteins. We used phosphorus magnetic resonance spectroscopy (P-31-MRS) to determine whether 17AAG treatment leads to alterations in phospholipids that could serve as pharmacodynamic markers for tumor response to 17AAG. Methods: HCT116, HT29, and SW620 colon cancer cells were treated with 17AAG, and extracts were examined by P-31-MRS. HT29 cells were also treated with the active metabolite of 17AAG, 17-amino,17- demethoxygeldanamycin (17AG), or the inactive 17AAG analog NSC683666. MF-1 nude mice carrying HT29 xenografts were examined using in vivo P-31-MRS before and after 17AAG treatment; xenograft. 3 tumor extracts were examined by P-31- MRS and proton MRS (H-1-MRS). Hsp90 client protein expression was determined by using western blots. Two-tailed t tests were used to compare metabolite concentrations and ratios, and a Mann-Whitney U test was used to compare proportions. All statistical tests were two-sided. Results: 17AAG treatment led to statistically significantly increased phosphocholine levels in all three cell lines (P =.02). 17AG treatment also increased phosphocholine levels in HT29 cells, whereas NSC683666 had no effect. The phosphomonoester/phosphodiester ratio was statistically significantly increased in the HT29 xenografts after 17AAG treatment relative to the pretreatment ratio (P =.02), whereas no statistically significant change was observed after vehicle treatment (P =.62). Statistically significant increases in phosphocholine, phosphoethanolamine, and valine levels were also observed in tumor extracts treated with 17AAG. Conclusions: Inhibition of Hsp90 by 17AAG resulted in altered phospholipid metabolism in cultured tumor cells and in tumor xenografts. The increases observed in phosphocholine and phosphomonoester levels suggest that these metabolites may have the potential to act as noninvasive pharmacodynamic markers for analyzing tumor response to treatment with 17AAG or other Hsp90 inhibitors.

Mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade glioma and secondary glioblastoma, represent an early pathogenic event, and are associated with epigenetically driven modulations of metabolism. Of particular interest is the recently uncovered relationship between the IDH1 mutation and decreased activity of the branched-chain amino acid transaminase 1 (BCAT1) enzyme. Noninvasive imaging methods that can assess BCAT1 activity could therefore improve detection of mutant IDH1 tumors and aid in developing and monitoring new targeted therapies. BCAT1 catalyzes the transamination of branched-chain amino acids while converting α-ketoglutarate (α-KG) to glutamate. Our goal was to use 13C magnetic resonance spectroscopy to probe the conversion of hyperpolarized [1-13C] α-KG to hyperpolarized [1-13C] glutamate as a readout of BCAT1 activity. We investigated two isogenic glioblastoma lines that differed only in their IDH1 status and performed experiments in live cells and in vivo in rat orthotopic tumors. Following injection of hyperpolarized [1-13C] α-KG, hyperpolarized [1-13C] glutamate production was detected both in cells and in vivo, and the level of hyperpolarized [1-13C] glutamate was significantly lower in mutant IDH1 cells and tumors compared with their IDH1-wild-type counterparts. Importantly however, in our cells the observed drop in hyperpolarized [1-13C] glutamate was likely mediated not only by a drop in BCAT1 activity, but also by reductions in aspartate transaminase and glutamate dehydrogenase activities, suggesting additional metabolic reprogramming at least in our model. Hyperpolarized [1-13C] glutamate could thus inform on multiple mutant IDH1-associated metabolic events that mediate reduced glutamate production. Cancer Res; 74(16); 4247–57. ©2014 AACR.

The prognosis for patients with pancreatic cancer is extremely poor, as evidenced by the disease's five-year survival rate of ~5%. New approaches are therefore urgently needed to improve detection, treatment, and monitoring of pancreatic cancer. MRS-detectable metabolic changes provide useful biomarkers for tumor detection and response-monitoring in other cancers. The goal of this study was to identify MRS-detectable biomarkers of pancreatic cancer that could enhance currently available imaging approaches. We used (1) H high-resolution magic angle spinning MRS to probe metabolite levels in pancreatic tissue samples from mouse models and patients. In mice, the levels of lipids dropped significantly in pancreata with lipopolysaccharide-induced inflammation, in pancreata with pre-cancerous metaplasia (4 week old p48-Cre;LSL-Kras(G12D) mice), and in pancreata with pancreatic intraepithelial neoplasia, which precedes invasive pancreatic cancer (8 week old p48-Cre LSL-Kras(G12D) mice), to 26 ± 19% (p = 0.03), 19 ± 16% (p = 0.04), and 26 ± 10% (p = 0.05) of controls, respectively. Lactate and taurine remained unchanged in inflammation and in pre-cancerous metaplasia but increased significantly in pancreatic intraepithelial neoplasia to 266 ± 61% (p = 0.0001) and 999 ± 174% (p0.00001) of controls, respectively. Importantly, analysis of patient biopsies was consistent with the mouse findings. Lipids dropped in pancreatitis and in invasive cancer biopsies to 29 ± 15% (p = 0.01) and 26 ± 38% (p = 0.02) of normal tissue. In addition, lactate and taurine levels remained unchanged in inflammation but rose in tumor samples to 244 ± 155% (p = 0.02) and 188 ± 67% (p = 0.02), respectively, compared with normal tissue. Based on these findings, we propose that a drop in lipid levels could serve to inform on pancreatitis and cancer-associated inflammation, whereas elevated lactate and taurine could serve to identify the presence of pancreatic intraepithelial neoplasia and invasive tumor. Our findings may help enhance current imaging methods to improve early pancreatic cancer detection and monitoring.

Summary Cancer cells must integrate multiple biosynthetic demands to drive indefinite proliferation. How these key cellular processes, such as metabolism and protein synthesis, crosstalk to fuel cancer cell growth is unknown. Here, we uncover the mechanism by which the Myc oncogene coordinates the production of the two most abundant classes of cellular macromolecules, proteins, and nucleic acids in cancer cells. We find that a single rate-limiting enzyme, phosphoribosyl-pyrophosphate synthetase 2 (PRPS2), promotes increased nucleotide biosynthesis in Myc-transformed cells. Remarkably, Prps2 couples protein and nucleotide biosynthesis through a specialized cis -regulatory element within the Prps2 5′ UTR, which is controlled by the oncogene and translation initiation factor eIF4E downstream Myc activation. We demonstrate with a Prps2 knockout mouse that the nexus between protein and nucleotide biosynthesis controlled by PRPS2 is crucial for Myc-driven tumorigenesis. Together, these studies identify a translationally anchored anabolic circuit critical for cancer cell survival and an unexpected vulnerability for "undruggable" oncogenes, such as Myc. PaperFlick

Deregulation of oncogenes and tumor suppressor genes is known to affect the expression and activity of transporters, sensors, and enzymes that regulate metabolism,1 but actual mutations in genes directly involved in metabolism, such as fumarate hydratase and succinate dehydrogenase, which are members of the tricarboxylic acid cycle, have only recently been demonstrated.2 Not surprisingly, then, the discovery of mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2), first in colon cancer and soon thereafter in different subtypes of gliomas, including oligodendrogliomas, astrocytomas, mixed oligoastrocytomas, and secondary glioblastomas,3,4 ignited a feverish interest in cancer cell metabolism. Initially, IDH1/2 mutations were anticipated to directly impact cellular metabolism and promote aerobic glycolysis, the metabolic pathway favored by cancer cells to support rapid proliferation.5,6 However, despite advances in the understanding of the function of the mutant IDH enzyme,7–10 the hypothesis that IDH1/2 mutations directly alter cancer cell metabolism remains unproven. 2-Hydroxyglutarate (2-HG), the product of the neomorphic activity of the mutant IDH1/2 enzymes, competitively inhibits α-ketoglutarate–dependent enzymes,11 including histone demethylases and 5-methylcytosine hydroxylases that are essential for epigenetic regulation of gene expression.12–14 Consequently, via 2-HG–dependent epigenetic remodeling, IDH mutations may interfere with differentiation, proliferation, survival, and metabolism.6 2-Hydroxyglutarate also affects the activity of EGLN prolyl 4-hydroxylases (PHDs), which are responsible for oxygen-dependent degradation of hypoxia-inducible factor (HIF)1α. Although stabilization of HIF1α by 2-HG has been reported,15 a recent study has clarified that 2-HG stimulates PHD activity, leading to the degradation of HIF1α.16 The subsequent downregulation of HIF1α-responsive genes by 2-HG may have unique consequences for human cancer cells, including limitation of the metabolic shift toward aerobic glycolysis, the so-called Warburg effect. A key step in glycolysis is the conversion of pyruvate to lactate, catalyzed by the lactate dehydrogenase (LDH) complex, one subunit of which is LDHA. LDHA enhances the efficiency of the LDH complex, allowing the rapid flux through glycolysis that is necessary to meet the energy needs of rapidly proliferating cells. As such, LDHA is considered a major biomarker of glycolytic activity.17–20 LDHA contains HIF1α binding sites in its promoter and is induced under hypoxic conditions, allowing normal cells to switch to an oxygen-independent glycolytic metabolic phenotype when deprived of oxygen.21,22 LDHA is overexpressed in cancer cells, and silencing of LDHA typically results in accelerated oxygen consumption, increased apoptosis, decreased proliferation, and strong inhibition of tumorigenicity.23–27 Here, we report that multiple HIF1α-responsive genes necessary for glycolysis are underexpressed in IDH mutant (mt) gliomas and brain tumor stem cells (BTSCs) derived from IDHmt tumors, including human BTSCs that have lost the mutant IDH1 allele and no longer produce 2-HG. Due to its central role in glycolysis, we focused our attention on LDHA. Our data suggest that LDHA is silenced through IDHmt-dependent methylation of its promoter. Silencing of LDHA and downregulation of other glycolytic genes is a surprising finding in the context of human cancer cells but may help to explain the slower progression and better prognosis of IDHmt gliomas.

Apoptosis was induced by treating L1210 leukaemia cells with mechlorethamine, and SW620 colorectal cells with doxorubicin. The onset and progression of apoptosis were monitored by assessing caspase activation, mitochondrial transmembrane potential, phosphatidylserine externalization, DNA fragmentation and cell morphology. In parallel, 31P magnetic resonance (MR) spectra of cell extracts were recorded. In L1210 cells, caspase activation was detected at 4 h. By 3 h, the MR spectra showed a steady decrease in NTP and NAD, and a significant build-up of fructose 1,6-bisphosphate (F-1,6-P) dihydroxyacetonephosphate and glycerol-3-phosphate, indicating modulation of glycolysis. Treatment with iodoacetate also induced a build-up of F-1,6-P, while preincubation with two poly(ADP-ribose) polymerase inhibitors, 3-aminobenzamide and nicotinamide, prevented the drop in NAD and the build-up of glycolytic intermediates. This suggested that our results were due to inhibition of glyceraldehyde-3-phosphate dehydrogenase, possibly as a consequence of NAD depletion following poly(ADP-ribose) polymerase activation. Doxorubicin treatment of the adherent SW620 cells caused cells committed to apoptosis to detach. F-1,6-P was observed in detached cells, but not in treated cells that remained attached. This indicated that our observations were not cell line- or treatment-specific, but were correlated with the appearance of apoptotic cells following drug treatment. The 31P MR spectrum of tumours responding to chemotherapy could be modulated by similar effects. © 1999 Cancer Research Campaign

A review of the literature has shown that in human breast tumours, large signals from phosphomonoesters (PME) and phosphodiesters (PDE) are evident. In serial measurements in 19 patients with breast cancer, a decrease in PME was significantly associated with a stable or responding disease (p = 0.017), and an increase in PME was associated with disease progression. Extract studies have shown PME to comprise of phosphoethanolamine (PEth) and phosphocholine (PCho), with the PEth to PCho ratio ranging from 1.3 to 12. The PCho content of high grade tumours was found to be higher than low grade tumours. In some animal models, changes in PCho have been shown to correlate with indices of cellular proliferation, and spheroid studies have shown a decrease in PCho content in spheroids with smaller growth fractions. A serial study of 25 patients with advanced primary breast tumours undergoing hormone, chemotherapy or radiotherapy treatments, showed that in this heterogenous group there were significant changes in metabolites that were seen during the first 3 weeks (range 2-4 weeks) of treatment, that correlated with volume change over this period, employed here as a measure of response. Changes in PME (p = 0.003), total phosphate (TP) (p = 0.008) and total nucleoside tri-phosphate (TNTP) (p = 0.02) over 3 (1) weeks were significantly associated with response, as were the levels of PME (p

Purpose: To identify mediators of glioblastoma antiangiogenic therapy resistance and target these mediators in xenografts. Experimental Design: We conducted microarray analysis comparing bevacizumab-resistant glioblastomas (BRG) with pretreatment tumors from the same patients. We established novel xenograft models of antiangiogenic therapy resistance to target candidate resistance mediator(s). Results: BRG microarray analysis revealed upregulation versus pretreatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pretreatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met–activated focal adhesion kinase and STAT3. We developed 2 novel xenograft models of antiangiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pretreatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met short hairpin RNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts. Conclusion: These findings support the role of c-Met in survival in hypoxia and invasion, features associated with antiangiogenic therapy resistance, and growth and therapeutic resistance of xenografts resistant to antiangiogenic therapy. Therapeutically targeting c-Met could prevent or overcome antiangiogenic therapy resistance. Clin Cancer Res; 19(7); 1773–83. ©2012 AACR.

Normal tissues express the M1 isoform of pyruvate kinase (PK) that helps generate and funnel pyruvate into the mitochondria for ATP production. Tumors, in contrast, express the less active PKM2 isoform, which limits pyruvate production and spares glycolytic intermediates for the generation of macromolecules needed for proliferation. Although high PKM2 expression and low PK activity are considered defining features of tumors, very little is known about how PKM expression and PK activity change along the continuum from low grade to high grade tumors, and how these changes relate to tumor growth. To address this issue, we measured PKM isoform expression and PK activity in normal brain, neural progenitor cells, and in a series of over 100 astrocytomas ranging from benign grade I pilocytic astrocytomas to highly aggressive grade IV glioblastoma multiforme (GBM). All glioma exhibited comparably reduced levels of PKM1 expression and PK activity relative to normal brain. In contrast, while grade I-III gliomas all had modestly increased levels of PKM2 RNA and protein expression relative to normal brain, GBM, regardless of whether they arose de novo or progressed from lower grade tumors, showed a 3-5 fold further increase in PKM2 RNA and protein expression. Low levels of PKM1 expression and PK activity were important for cell growth as PKM1 over-expression and the accompanying increases in PK activity slowed the growth of GBM cells. The increased expression of PKM2, however, was also important, because shRNA-mediated PKM2 knockdown decreased total PKM2 and the already low levels of PK activity, but paradoxically also limited cell growth in vitro and in vivo. These results show that pyruvate kinase M expression, but not pyruvate kinase activity, is regulated in a grade-specific manner in glioma, but that changes in both PK activity and PKM2 expression contribute to growth of GBM.

Temozolomide (TMZ) is a DNA methylating agent used to treat brain cancer. TMZ-induced O6-methylguanine adducts, in the absence of repair by O6-methylguanine DNA methyltransferase (MGMT), mispair during DNA replication and trigger cycles of futile mismatch repair (MMR). Futile MMR in turn leads to the formation of DNA single and double strand breaks, Chk1 and Chk2 phosphorylation/activation, cell cycle arrest, and ultimately cell death. Although both pChk1 and pChk2 are considered to be biomarkers of TMZ-induced DNA damage, cell-cycle arrest, and TMZ induced cytotoxicity, we found that levels of pChk1 (ser345), its downstream target pCdc25C (ser216), and the activity of its upstream activator ATR, were elevated within 3 hours of TMZ exposure, long before the onset of TMZ-induced DNA double strand breaks, Chk2 phosphorylation/activation, and cell cycle arrest. Furthermore, TMZ-induced early phosphorylation of Chk1 was noted in glioma cells regardless of whether they were MGMT-proficient or MGMT-deficient, and regardless of their MMR status. Early Chk1 phosphorylation was not associated with TMZ-induced reactive oxygen species, but was temporally associated with TMZ-induced alkalai-labile DNA damage produced by the non-O6-methylguanine DNA adducts and which, like Chk1 phosphorylation, was transient in MGMT-proficient cells but persistent in MGMT-deficient cells. These results re-define the TMZ-induced DNA damage response, and show that Chk1 phosphorylation is driven by TMZ-induced mismatch repair-independent DNA damage independently of DNA double strand breaks, Chk2 activation, and cell cycle arrest, and as such is a suboptimal biomarker of TMZ-induced drug action.

To optimize the development of stem cell (SC)-based therapies for the treatment of glioblastoma (GBM), we compared the pathotropism of 2 SC sources, human mesenchymal stem cells (hMSCs) and fetal neural stem cells (fNSCs), toward 2 orthotopic GBM models, circumscribed U87vIII and highly infiltrative GBM26. High resolution and contrast-enhanced (CE) magnetic resonance imaging (MRI) were performed at 14.1 Tesla to longitudinally monitor the in vivo location of hMSCs and fNSCs labeled with the same amount of micron-size particles of iron oxide (MPIO). To assess pathotropism, SCs were injected in the contralateral hemisphere of U87vIII tumor-bearing mice. Both MPIO-labeled SC types exhibited tropism to tumors, first localizing at the tumor edges, then in the tumor masses. MPIO-labeled hMSCs and fNSCs were also injected intratumorally in mice with U87vIII or GBM26 tumors to assess their biodistribution. Both SC types distributed throughout the tumor in both GBM models. Of interest, in the U87vIII model, areas of hyposignal colocalized first with the enhancing regions (ie, regions of high vascular permeability), consistent with SC tropism to vascular endothelial growth factor. In the GBM26 model, no rim of hyposignal was observed, consistent with the infiltrative nature of this tumor. Quantitative analysis of the index of dispersion confirmed that both MPIO-labeled SC types longitudinally distribute inside the tumor masses after intratumoral injection. Histological studies confirmed the MRI results. In summary, our results indicate that hMSCs and fNSCs exhibit similar properties regarding tumor tropism and intratumoral dissemination, highlighting the potential of these 2 SC sources as adequate candidates for SC-based therapies.

Alterations in cell metabolism are increasingly being recognized as a hallmark of cancer and are being exploited for the development of diagnostic tools and targeted therapeutics. Recently, ¹³C MRS-detectable hyperpolarized pyruvate to lactate conversion has been validated in models as a noninvasive imaging method for the detection of tumors and treatment response, and has successfully passed phase I clinical trials. To date, response to treatment has been associated with a decrease in hyperpolarized lactate production. In this study, we monitored the effect of treatment with the mitogen-activated protein kinase (MEK) inhibitor U0126 in prostate and breast cancer cells. Following treatment, we observed a 31% decrease in the flux of hyperpolarized ¹³C label in treated MCF-7 breast cancer cells relative to controls. In contrast, and unexpectedly, the flux increased to 167% in treated PC3 prostate cancer cells. To mechanistically explain these observations, we investigated treatment-induced changes in the different factors known to affect the pyruvate to lactate conversion. NADH (nicotinamide adenine dinucleotide, reduced form) levels remained unchanged, whereas lactate dehydrogenase expression and activity, as well as intracellular lactate, increased in both cell lines, providing an explanation for the elevated hyperpolarized lactate observed in PC3 cells. The expression of MCT1, which mediates pyruvate transport, decreased in treated MCF-7, but not PC3, cells. This identifies pyruvate transport as rate limiting in U0126-treated MCF-7 cells and explains the decrease in hyperpolarized lactate observed in these cells following treatment. Our findings highlight the complexity of interactions between MEK and metabolism, and the need for mechanistic validation before hyperpolarized ¹³C MRS can be used to monitor treatment-induced molecular responses.

Somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in 70-90% of low-grade gliomas. The mutation leads to the production of 2-hydroxyglutarate (2-HG), a novel “oncometabolite” that alters the epigenetic profile of DNA and inhibits cellular differentiation. Recent studies indicate that metabolic changes in IDH1 mutant cells extend beyond 2-HG production. We recently demonstrated that steady state levels of glutamate, lactate and phosphocholine were reduced in IDH1 mutant cells using two genetically engineered models, a U87 glioblastoma-based model and a normal human astrocyte (NHA) model. In this study, we explored the mechanism behind the reduction in glutamate levels in IDH1 mutant cells. We discovered that pyruvate dehydrogenase (PDH) activity was significantly reduced in U87 and NHA IDH1 mutant cells compared to wild-type cells. Reduced PDH activity was associated with increased inhibitory phosphorylation of PDH in both models. Examination of mRNA and protein levels for the pyruvate dehydrogenase kinases (PDKs) revealed that PDK3 expression was up-regulated in IDH1 mutant cells in both models. Levels of hypoxia inducible factor-1α (HIF-1α), an established regulator of PDK3 expression in cancer cells, was also higher in IDH1 mutant cells. Importantly, treatment of U87 and NHA IDH1 wild-type cells with 2-HG recapitulated the effects of the IDH1 mutation, with higher levels of HIF-1α and PDK3, and reduced PDH activity, in 2-HG treated cells. Importantly, pharmacological activation of PDH activity by treatment with dichloroacetate, a PDK inhibitor, completely abrogated the increase in clonogenicity conferred by the IDH1 mutation. Treatment of patient-derived mutant IDH1 neurosphere models (BT142 and BT54 cell lines) with dichloroacetate further confirmed that PDH activity is essential for cell proliferation. Thus, our results indicate that reversing the down regulation of PDH activity in IDH1 mutant glioma cells adversely affects their proliferation, a finding with therapeutic implications.